www.jax.org

Optogenetic, transient-sensing and chemogenetic mouse models available from The Jackson Laboratory.

ABSTRACT Advancing the understanding of neural circuitry in both normal and diseased states is a priority of the biomedical community. To facilitate this challenge, The Jackson Laboratory (JAX) Mouse Repository offers an impressive array of genetically engineered tools enabling scientists to monitor neuronal activity in the intact mouse brain. Top most in this tool box are mouse lines with optogenetic and transient-sensing (calcium-, voltage-, glutamate-) technologies. Opsins are light-activated proteins that alter membrane potential in neurons, so that stimulation with light allows rapid control of neuronal activity. Several mouse lines express improved/optimized opsins fused to fluorescent proteins. These include mice with channelrhodopsin expression directed by specific promoters. Additional control is available in mice with Cre- or Tet-dependent expression of channelrhodopsin or halorhodopsin. Variants of GCaMP fluoresce in response to calcium binding and serve as an indicator of neuronal activation. These include Thy1-promoter driven GCaMP6 transgenic lines, Tet-dependent GCaMP6f or GCaMP6s transgenic lines and Cre-dependent GCaMP3, GCaMP5, GCaMP6f or GCaMP6s mouse lines. Several strains utilize both Cre-lox and Tet-On/-Off functionality. Removal of a floxed-STOP allows Tet-dependent expression of channelrhodopsin (Chronos/EGFP), halorhodopsin (Jaws/EGFP), GCaMP6s, GCaMP6f or glutamate-sensor (iGluSnFR). New models have inducible expression of the QuasAr voltage-indicator combined with the CheRiff channelrhodopsin (OptoPatch) or the GCaMP6f calcium sensor (CaViAr). This set includes mice created by the Allen Institute for Brain Science, the Genetically-Encoded Neuronal Indicator and Effector (GENIE) Project (Janelia/HHMI), Duke/MIT and several others. Designer receptors exclusively activated by designer drugs (DREADDs) are mutant G-protein coupled receptors activated by the pharmacologically-inert molecule clozapine-N-oxide. Similarly, a glycine-insensitive mutant human glycine α1 receptor induces a hyperpolarizing chloride current following ivermectin administration. Several chemogenetic strains have Cre-, Tet- and/or FLP-inducible expression of M3Dq, M4Di, M3Ds or GlyClαF207A/A288G. The JAX Mouse Repository receives support from NIH, HHMI and private foundations.

Search The Jackson Laboratory Repository / JAX® Mice Database www.jax.org/mouse-search

The Jackson Laboratory Resources for Optogenetics, Cre-dependent Optogenetic

Tools and Cre Strains for Neurobiology www.jax.org/optogenetics

www.jax.org/donate-a-mouse Donating a Strain to

The Jackson Laboratory

OPTOGENETICS A. INTRODUCTION

OPTOGENETICS: control of cellular functions in genetically modified cells using opsins - transmembrane, retinal-binding proteins that combine a light-sensitive domain with an ion channel or pump. Upon absorption of light, the protein is activated and provides ion transport, membrane potential alteration and sensory functions to bacteria. By exogenously expressing light-activated proteins that alter membrane potential in neurons, addition or removal of specific wavelengths of light can be used for rapid control of neuronal activity.

B. OPSINS

C. DESIGN FOR GENETICALLY-ENCODED OPTOGENETIC MODELS Create a targeted mutation or transgene in mouse. Often, a fluorescent protein is engineered to be co-expressed.

D. APPLICATIONS

Fiber optic electrode delivers specific wavelengths of light = opens the ion channel = light controls neuronal signaling

Archaerhodopsin-3 (Arch)

outward proton pump hyperpolarizes cell -

prevents action potentials in response to

yellow-green light

Halorhodopsin (NpHR)

inward chloride ion pump hyperpolarizes cell -

prevents action potentials in response to

yellow light

Channelrhodopsin-2 (ChR2)

cation channel depolarizes cell -

causes action potentials in response to

blue light.

Images from Karl Deisseroth Optogenetics website, Williams and Deisseroth 2013 PNAS

110:16287 and Häusser 2014 Nat Methods 11:1012

PROMOTER STOP loxP EFFECTOR loxP

PROMOTER EFFECTOR COMMON NAME JAX#

R26 :: CAG Arch / GFP Ai35D 012735 R26 :: CAG ArchT / EGFP Ai40D 021188 R26 :: CAG ChETA/ChR2*H134R ; tdT 2xChETA-tdT 017455 R26 :: CAG ChR2*H134R / EYFP Ai32 024109 R26 :: CAG hChR2*H134R / tdT Ai27D 012567 R26 :: CAG ReaChR / mCitrine Rosa26 CAG-LSL-ReaChR-mCit 026294 R26 :: CAG eNpHR3.0 / EYFP Ai39 014539 R26 :: CAG GCaMP3 Ai38 014538

RNA pol II :: CAG GCaMP5G PC::G5-tdT 024477 R26 :: CAG GCaMP6f [membrane] R26-Lck-GCaMP6f flox 029626 R26 :: CAG GCaMP6f Ai95D (C57BL/6J) 028865 R26 :: CAG GCaMP6s Ai96 (C57BL/6J) 028866 R26 :: CAG mOrange2 ; EGFP ; mKate2 RGBow reporter 028583 R26 :: CAG Synaptophysin / tdT Ai34D 012570 R26 :: CAG hVOS1.5 voltage sensor Ai35-hVOS1.5 031102

Cre-Inducible Lines A floxed-STOP cassette prevents transcription of opsin/xFPs,

calcium sensors, photoactivatable GFP, etc.

Cre:Tet-Inducible Lines

TIGRE locus (Igs7) ~21.54 Mbp on Chr.9 TREtight STOP loxP EFFECTOR loxP

Specific Promoters drive expression of opsin/xFPs,

Ca+ sensors and photoactivatable GFP

Thy1-GCaMP6s Thy1-GCaMP6f Line GP4.3 GP4.12 GP5.1 GP5.5 GP5.11 GP5.17 JAX# 024275 025776 028279 024276 024339 025393

Olf. bulb + ++ ++ ++ ++ ++ M1 ++ +++ ++ ++ ++ +++

Piriform + ++ ++ + + + Amygdala + +++ ++ ++ + +++

S1 ++ +++ ++ ++ ++ +++ Hippocamp +++ +++ +++ +++ ++ +++ Thalamus + + + + + + Hypothal - + + + + +

V1 ++ ++ ++ ++ ++ +++ Cerebellum - - + - + -

Midbrain + + + - - + Pons + + + + + ++

Medulla + - - - - +

Thy1-GCaMP6 Lines

Founder line-specific expression of GCaMP6 variants in brain from Dana et al. 2014 PLoS

One. 9:e108697

Table 1.

Following Cre recombination, mice have dox-inducible / reversible

expression of opsins, opsin/xFPs and calcium and glutamate sensors

Program/Poster#:

809.04 / VV46 Jason Beckwith, Stephen F. Rockwood, Cathleen Lutz The Jackson Laboratory, Bar Harbor, Maine 04609 USA

TRANSIENT-SENSORS

• Ca2+ binding (neuron activation) EGFP fluorescence (~510 nm) • Optimized dynamic range, baseline fluorescence, sensitivity & function

GCaMP6s : ultrasensitive with slower decay & response kinetics GCaMP6f : fast response kinetics

GCaMP illustration adapted from Akerboom et al. 2012 J Neurosci 32:13819

Calcium Indicators : GCaMP

Voltage Indicators : OptoPatch and CaViAr

PROMOTER EFFECTOR EXPRESSION NAME JAX#

TRE hM3Dq Tet- and CNO-inducible Gq TRE-hM3Dq 014093

R26 :: CAG hM3Dq Cre-inducible mCitrine ; then CNO-inducible Gq

R26-LSL-Gq-DREADD 026220

R26 :: CAG hM3Dq FLP-inducible eGFP,; then Cre-inducible mCherry & CNO-inducible Gq RC::FL-hM3Dq 026942

R26 :: CAG hM3Dq Cre-inducible mCherry ; then CNO-inducible Gq RC::L-hM3Dq 026943

TRE hM4Di Tet- and CNO-inducible Gi TRE-hM4Di 024114

R26 :: CAG hM4Di Cre-inducible mCitrine ; then CNO-inducible Gi

R26-LSL-Gi-DREADD 026219

R26 :: CAG hM4Di FLP-inducible mCherry ; then Cre- & CNO-inducible Gi RC::FPDi 029040

TRE GlyClαF207A/A288G Tet- & IVM-inducible GlyCl channel :: eYFP TRE-GlyCl 029301

CHEMOGENETICS DREADD

CheRiff : sensitive blue light-activated ChR variant; fused to eGFP

QuasAr2: near infrared-activated, Arch-derived, enhanced voltage indicator; fused to a membrane-targeted dark Orange2

QuasAr3: improved in vivo trafficking; fused to membrane-targeted Citrine

CaViAr GCaMP6f::QuasAr2-mOr2

Allows simultaneous Ca2+ and

voltage imaging

OptoPatch2 / OptoPatch3 CheRiff-eGFP::QuasAr-mxFP

Allows simultaneous, all-optical

electrophysiology

“Designer Receptors Exclusively Activated by Designer Drugs” are mutant G protein-coupled receptors activated by CNO.

hM3Dq: activation Gq-DREADD (burst firing) hM4Di: inhibitory Gi-DREADD (neuronal silencing) rM3Ds: activation Gs-DREADD (increase cAMP).

Gly Chloride Channels Induce hyperpolarizing Cl current via ivermectin (IVM).

GlyCl: glycine-insensitive GlyClαF207A/A288G receptor

PROM EFFECTOR EXPRESSION NAME JAX#

R26 :: CAG CheRiff-eGFP:: QuasAr2-mOrange2 Cre-inducible Floxopatch,

Optopatch2 028678

TIGRE :: TRE CheRiff-eGFP:: QuasAr2-mOrange2

Cre-inducible & Tet-control

Optopatch2 Ai141 029677

TIGRE :: TRE CheRiff-eGFP:: QuasAr3-mCitrine

Cre-inducible & Tet-control

Optopatch3 Ai155 029679

TIGRE :: TRE QuasAr2-mOrange2:: GCaMP6f [membrane]

Cre-inducible & Tet-control

CaViAr Ai142 029678

PROMOTER EFFECTOR COMMON NAME JAX# TIGRE :: TRE GCaMP6f Ai93D 024103

TIGRE :: TRE ; R26 GCaMP6f + tTA Ai93D;ROSA26-ZtTA 024107 TIGRE :: TRE ; CaMK2a GCaMP6f + tTA Ai93D;CaMK2a-tTA 024108 TIGRE :: TRE2 + CAG GCaMP6f + tTA2 Ai148D 030328

TIGRE :: TRE GCaMP6s Ai94D 024104 TIGRE :: TRE ; R26 GCaMP6fs + tTA Ai94D;ROSA26-ZtTA 024112

TIGRE :: TRE ; CaMK2a GCaMP6fs + tTA Ai94D;CaMK2a-tTA 024115 TIGRE :: TRE RCaMP1.07 Ai143D 030217 TIGRE :: TRE iGluSnFr Ai85D 026260 TIGRE :: TRE Jaws / EGFP (Halo57/ EGFP) Ai79D 023529 TIGRE :: TRE ReaChR / EYFP Ai136D 030216 TIGRE :: TRE ShChR (Chronos) Ai90D 024100 TIGRE :: TRE ssAPEX2tm Ai133D 030213

TIGRE :: TRE2 + CAG EGFP + tdT + tTA2 Ai139D 030219 TIGRE :: TRE2 + CAG EGFP + tTA2 Ai140D 030220

PROMOTER EFFECTOR EXPRESSION COMMON NAME JAX#

human ubiquitin C PA-GFP widespread UBC PA-GFP 022486 thymus cell antigen 1 ChR2 / EYFP widespread brain Thy1-ChR2-EYFP

line 18 007612 thymus cell antigen 1 ChR2 / EYFP widespread brain Thy1-ChR2-EYFP

line 9 007615

parvalbumin mhChR2 / EYFP PV+ GABAergic interneurons

PV-hChR2(H134R)-EYFP line 15 012355

choline acetyltransferase mhChR2 / EYFP cholinergic

neurons ChAT-ChR2(H134R)-EYFP

line 6 014546 vesicular GABA

transporter mhChR2 / EYFP GABAergic interneurons VGAT-ChR2(H134R)-EYFP 014548

olfactory receptor 160 ChR2*H134R / EYFP M72+ olfactory sensory

neurons M72-IRES-ChR2-YFP 021206 thymus cell antigen 1 GCaMP3 widespread brain Thy1-GCaMP3 line 6

(C57BL/6) 029860

TRE GCaMP6s Tet-inducible TRE-GCaMP6s line G6s2 024742 synaptosomal-

assoc. protein 25 GCaMP6s widespread brain Snap25-2A-GCaMP6s-D 025111

CAG GECO1.2 / mCherry primary cilia hArl13b-mCherry-GECO1.2tg 030613

thymus cell antigen 1 jRGECO1a brain (denser cortex) Thy1-jRGECO1a-WPRE

line GP8.20 030525 thymus cell antigen 1 jRGECO1a brain (sparser cortex) Thy1-jRGECO1a-WPRE

line GP8.31 030526 thymus cell antigen 1 jRGECO1a brain Thy1-jRGECO1a-WPRE

line GP8.58 030527 thymus cell antigen 1 jRGECO1a brain Thy1-jRGECO1a-WPRE

line GP8.62 030528

Mutant Mouse Resource and Research Center at JAX Human APP / PS1 mice with Alzheimer’s disease mutations

MMRRC# COMMON NAME Donated By 034830-JAX 3xTg-AD Dr. Frank LaFerla (Univ of California, Irvine) 034840-JAX 5xFAD Dr. Robert Vassar (Northwestern University) 034829-JAX APPswe/PSEN1dE9 Dr. David Borchelt (Univ of Florida)

PROMOTER EFFECTOR or xFP loxP STOP loxP STOP

Cre:FLP Dual Inducible Lines Multiple STOP cassettes prevent transcription of

opsin/xFPs or fluorescent protein.

PROMOTER EFFECTOR STOPS COMMON NAME JAX#

R26 :: CAG CatCh / EYFP (ChR2L132C / EYFP)

frt-STOP-frt :: lox-STOP-lox Ai80D 025109

R26 :: CAG ReaChR / mCitrine frt-STOP-frt :: lox-STOP-lox

Rosa26 CAG-FSF-LSL-ReaChR-mCit 024846

R26 :: CAG Synaptophysin / EGFP + tdTomato

frt-STOP-frt :: lox-STOP-lox RC::FPSit 030206

R26 :: CAG tdT frt-STOP-frt :: lox-STOP-lox Ai65D 021875

R26 :: CAG tdT ; EGFP frt-STOP-frt :: lox-tdT-lox RC::FLTG 026932 www.jax.org

Ai35-hVOS1.5 x Cre double mutant :: cells with expression x Parv-Cre x CalCrl,Cre x GAD2-Cre

x Calb2-CreERT2

x Fos-CreERT2

x Nestin-CreERT2

JAX#017320 JAX#023014 JAX#019022 JAX#013730 JAX#021882 JAX#016261 PV+

interneuron hilar mossy GABAergic inhibitory

calretinin interneuron

electrically-activated

newborn granule

FEATURED IMAGES using Ai35-hVOS1.5 (JAX#031102): Optimized hybrid voltage sensor probe expression in genetically-defined subpopulations.

Images courtesy Dr. Meyer Jackson from Bayguinov et al. 2017 J Neurosci 37:9305.

FEATURED IMAGES: Cortical imaging of Thy1-jRGECO1a-WPRE lines GP8.20,

GP8.31, GP8.58, GP8.62 (JAX#s 030525 030526 030527 030528) [left-to-right].

Images courtesy Drs. Hod Dana and Douglas Kim.

FEATURED IMAGES: TRE-GlyCl in forebrain (via CaMKII-tTA) or

entorhinal cortex (via Klk8-tTA). See JAX#s 029301 and 007004.

Images courtesy of Dr. Joanna Jankowsky.